Magnetic Force-Based Microfluidic Techniques for Cellular and Tissue Bioengineering

PubMed: 30619842Live cell manipulation is an important biotechnological tool for cellular and tissue level bioengineering applications due to its capacity for guiding cells for separation, isolation, concentration, and patterning. Magnetic force-based cell manipulation methods offer several advantages, such as low adverse effects on cell viability and low interference with the cellular environment. Furthermore, magnetic-based operations can be readily combined with microfluidic principles by precisely allowing control over the spatiotemporal distribution of physical and chemical factors for cell manipulation. In this review, we present recent applications of magnetic force-based cell manipulation in cellular and tissue bioengineering with an emphasis on applications with microfluidic components. Following an introduction of the theoretical background of magnetic manipulation, components of magnetic force-based cell manipulation systems are described. Thereafter, different applications, including separation of certain cell fractions, enrichment of rare cells, and guidance of cells into specific macro- or micro-arrangements to mimic natural cell organization and function, are explained. Finally, we discuss the current challenges and limitations of magnetic cell manipulation technologies in microfluidic devices with an outlook on future developments in the field

Development and verification of a three-dimensional (3D) breast cancer tumor model composed of circulating tumor cell (CTC) subsets

Inevi, Muge Anil/0000-0003-2854-3472; Gulce Iz, Sultan/0000-0002-9275-6272; saglam metiner, pelin/0000-0002-5726-1928WOS: 000489549500001PubMed: 31583566Breast cancer is one of the most common cancer types among women in which early tumor invasion leads to metastases and death. EpCAM (epithelial cellular adhesion molecule) and HER2 (human epidermal growth factor receptor 2) are two main circulating tumor cell (CTC) subsets in HER2+ breast cancer patients. in this regard, the main aim of this study is to develop and characterize a three-dimensional (3D) breast cancer tumor model composed of CTC subsets to evaluate new therapeutic strategies and drugs. For this reason, EpCAM(+) and HER2(+) sub-populations were isolated from different cell lines to establish 3D tumor model that mimics in situ (in vivo) more closely than two-dimensional (2D) models. EpCAM(+)/HER2(+) cells had a high proliferation rate and low tendency to attach to the surface in comparison with parental MDA-MB-453 cells as CTC subsets. Aggressive breast cancer subpopulations cultured in 3D porous chitosan scaffold had enhanced cell-cell and cell-matrix interactions compared to 2D cultured cells and these 3D models showed more aggressive morphology and behavior, expressed higher levels of pluripotency marker genes, Nanog, Sox2 and Oct4. For the verification of the 3D model, the effects of doxorubicin which is a chemotherapeutic agent used in breast cancer treatment were examined and increased drug resistance was determined in 3D cultures. the 3D tumor model comprising EpCAM(+)/HER2(+) CTC subsets developed in this study has a promising potential to be used for investigation of an aggressive CTC microenvironment in vitro that mimics in vivo characteristics to test new drug candidates against CTCs.Ege UniversityEge University [15-MUH-038]; [TUBITAK-1919B011503631]This project was supported by the research fund as Ege University Scientific Research Project (Project Number: 15-MUH-038) and by TUBITAK-1919B011503631 under the supervision of Sultan GULCE-IZ. the authors would like to thank Dr Aylin SENDEMIR-URKMEZ for providing the chitosan scaffolds

A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells

WOS: 000419607700024PubMed ID: 28685239Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-alpha), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-alpha mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-alpha mAb production from the humanized anti-TNF-alpha mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-alpha production. The production of anti-TNF-alpha in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-alpha MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [2209-A, 1919B011402737]This study has been partly funded by TUBITAK through 2209-A project number 1919B011402737 under the supervision of Sultan GULCE IZ, PhD

Immunogenicity of a xenogeneic multi-epitope HER2(+) breast cancer DNA vaccine targeting the dendritic cell restricted antigen-uptake receptor DEC205

Breast cancer was ranked first in global cancer incidence in 2020, and HER2 overexpression in breast cancer accounts for 20-30% of breast cancer patients. Current therapeutic strategies increase the survival rate, but resistance to them occurs frequently, and there is an urgent need to develop novel treatments such as DNA vaccines which can induce a specific and long-lasting immune response against HER2 antigens. To enhance the immunogenicity of DNA vaccines, dendritic cells (DCs) can be targeted using multiepitope proteins that provide accurate immune focusing. For this purpose, we generated a DNA vaccine encoding a fusion protein composed of 1) in silico discovered antigenic epitopes of human and rat HER2 proteins (MeHer2) and 2) a single-chain antibody fragment (ScFv) specific for the DC-restricted antigen uptake receptor DEC205 (ScFvDEC). The xenogeneic multi-epitope DNA vaccine (pMeHer2) encodes three only T-cell epitopes, two only B-cell epitopes, and two T and B cell epitopes, and pScFvDEC-MeHer2 vaccine additionally encodes ScFvDEC introduced at the N terminus of the MeHer2. Then, mouse groups were immunized with pScFvDEC-MeHer2, pMeHer2, pScFvDEC, pEmpty, and PBS to determine the elicited immune response. pScFvDEC-MeHer2 vaccinated mice showed a strong IgG response (P < 0.0001) and pScFvDEC-MeHer2 induced a significant IgG2a increase (P < 0.01). The percentages of both IFN-gamma secreting CD4 and CD8 T cells were higher in mice immunized with pScFvDEC-MeHer2 compared with the pMeHer2. pScFvDEC-MeHer2 and pMeHer2 secreted significantly higher levels of extracellular IFN-gamma compared with to control groups (P < 0.0001). In addition, the IFN-gamma level of the pScFvDEC-MeHer2 vaccine group was approximately two times higher than the pMeHer2 group (P < 0.0001). Overall, this study identified the pScFvDECMeHer2 construct as a potential DNA vaccine candidate, supporting further studies to be conducted on HER2(+)& nbsp;animal models.(c) 2022 Elsevier Ltd. All rights reserved.Scientific Research Projects Branch Directorate of Ege University, Turkey [2016-TIP-082]Acknowledgement This study was supported by the grant given by the Scientific Research Projects Branch Directorate of Ege University, Turkey (Grant No: 2016-TIP-082) to L.Y

μDACS platform: A hybrid microfluidic platform using magnetic levitation technique and integrating magnetic, gravitational, and drag forces for density-based rare cancer cell sorting

Circulating tumor cells (CTCs) are crucial indicators of cancer metastasis. However, their rarity in the bloodstream and the heterogeneity of their surface biomarkers present challenges for their isolation. Here, we developed a hybrid microfluidic platform (microfluidic-based density-associated cell sorting (µDACS) platform) that utilizes density as a biophysical marker to sort cancer cells from the population of white blood cells (WBCs). The platform utilizes the magnetic levitation technique on a microfluidic chip to sort cells based on their specific density ranges, operating under a continuous flow condition. By harnessing magnetic, gravitational, and drag forces, the platform efficiently separates cells. This approach involves a microfluidic chip equipped with a microseparator, which directs cells into top and bottom outlets depending on their levitation heights, which are inversely proportional to their densities. Hence, low-density cancer cells are collected from the top outlet, while high-density WBCs are collected from the bottom outlet. We optimized the sorting efficiency by varying the flow rates, and concentrations of the sorting medium's paramagnetic properties using standard densities of polymeric microspheres. To demonstrate the platform's applicability, we performed hybrid microfluidic sorting on MDA-MB-231 human breast cancer cells and U-937 human monocytes. The results showed efficient sorting of rare cancer cells (≥100 cells/mL) from serum samples, achieving a sorting efficiency of ∼70% at a fast-processing speed of 1 mL h−1. This label-free approach holds promise for rapid and cost-effective CTC sorting, facilitating in-vitro diagnosis and prognosis of cancer